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Evolving a 24-hr oscillator in budding yeast.

Wildenberg GA, Murray AW - Elife (2014)

Bottom Line: We identified eight putative causative mutations in one clone and recreated the evolved phenotype in the ancestral strain.The mutated genes lack obvious relationships to each other, but multiple lineages change from the haploid to the diploid pattern of gene expression.We show that a novel, complex phenotype can evolve by small sets of mutations in genes whose molecular functions appear to be unrelated to each other.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, United States.

ABSTRACT
We asked how a new, complex trait evolves by selecting for diurnal oscillations in the budding yeast, Saccharomyces cerevisiae. We expressed yellow fluorescent protein (YFP) from a yeast promoter and selected for a regular alternation between low and high fluorescence over a 24-hr period. This selection produced changes in cell adhesion rather than YFP expression: clonal populations oscillated between single cells and multicellular clumps. The oscillations are not a response to environmental cues and continue for at least three cycles in a constant environment. We identified eight putative causative mutations in one clone and recreated the evolved phenotype in the ancestral strain. The mutated genes lack obvious relationships to each other, but multiple lineages change from the haploid to the diploid pattern of gene expression. We show that a novel, complex phenotype can evolve by small sets of mutations in genes whose molecular functions appear to be unrelated to each other.

No MeSH data available.


Related in: MedlinePlus

Dynamics of oscillator aggregate assembly.(A) Low magnification images of synchronized E6-1 cultures that contained two subclones, one labeled with CFP (CFP+) and one without CFP (CFP−). Single cells that were selected as dim events by FACS and whose cell walls were labeled with Oregon green 488-X assemble a lineage-based clump and do not adhere to other cell lineages (i.e., no CFP (+/−) clumps) (left panels). At the peak multicellular phase, clumps were labeled with Oregon green 488-X and followed. These cells give rise to the single cell stage by fragmenting and producing non-adherent daughters so that the majority of the population is single celled by the end of the complete, 24-hr cycle (right panels). (B) Schematic of the mixing experiment performed by mixing, sonicating, and incubating asynchronous CFP+ and CFP− populations: cell separation defects will result in single colored clumps but adhesive cells will stick non-specifically to each other and produce clumps containing cells of both colors. Representative image of a single clump (white outline) showing E6-1 cells form clumps by adhering to each other.DOI:http://dx.doi.org/10.7554/eLife.04875.006
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fig2s1: Dynamics of oscillator aggregate assembly.(A) Low magnification images of synchronized E6-1 cultures that contained two subclones, one labeled with CFP (CFP+) and one without CFP (CFP−). Single cells that were selected as dim events by FACS and whose cell walls were labeled with Oregon green 488-X assemble a lineage-based clump and do not adhere to other cell lineages (i.e., no CFP (+/−) clumps) (left panels). At the peak multicellular phase, clumps were labeled with Oregon green 488-X and followed. These cells give rise to the single cell stage by fragmenting and producing non-adherent daughters so that the majority of the population is single celled by the end of the complete, 24-hr cycle (right panels). (B) Schematic of the mixing experiment performed by mixing, sonicating, and incubating asynchronous CFP+ and CFP− populations: cell separation defects will result in single colored clumps but adhesive cells will stick non-specifically to each other and produce clumps containing cells of both colors. Representative image of a single clump (white outline) showing E6-1 cells form clumps by adhering to each other.DOI:http://dx.doi.org/10.7554/eLife.04875.006

Mentions: To analyze the oscillations in cell association, we followed the behavior of E6-1 cells grown at low population density using two different labels. The first, cerulean fluorescent protein (CFP) was expressed from a constitutive promoter (PACT1) to distinguish two genetically identical subclones: one expressed CFP (CFP+) and the other did not (CFP−). The second was introduced by labeling cells, just after FACS selection, by covalently linking Oregon Green 488-X, a yellow fluorescent molecule, to their cell walls (Hoch et al., 2005). Because a daughter’s cell wall is entirely new, the original cells retain the yellow label and their daughters are unlabeled (Barral et al., 2000). We followed these cultures as they proliferated and eventually switched to the other phase of the morphological oscillation (Figure 2A,B and Figure 2—figure supplement 1A). Single cells that were selected for by sorting for low YFP fluorescence and covalently labeled (T = 0 hr), proliferated to become clumps composed of a single yellow cell surrounded with non-yellow cells that had the same CFP expression state as the original single cell. The absence of clumps containing more than one yellow cell or both CFP+ and CFP− cells in the same clump shows that cells emerging from the single-cell phase build lineage-based clumps. Consistent with this interpretation, we saw synchronous oscillations in cell association even at very high dilution (∼103 cells/ml). Multicellular clumps, which were covalently labeled at the time of peak aggregation gave rise to single cells by a combination of two mechanisms: fragmenting to produce smaller clumps and proliferating to produce single-celled offspring. Smaller clumps consisting of yellow cells and newly born non-yellow cells were observed 3 hr after (T = 13 hr) the peak aggregation time (10 hr). By 18 hr, the population was a mixture of medium-sized and smaller clumps and single cells, and by 24 hr non-yellow, single cells, and small clumps were the bulk of the population, representing the completion of one cycle. When unsynchronized CFP+ and CFP− subclones were sonicated to break up all the clumps and cultured together for 90 min, clumps that re-associated were composed of a mixture of CFP+ and CFP− cells showing that clumps form by cells sticking together rather than failing to separate after division (Figure 2—figure supplement 1B). These results show that cells oscillate between two states over a 24-hr period: single cells produce offspring that immediately stick to their mothers to assemble a lineage-based clump, which later produces a population of single cells and small clumps as a result of clumps fragmenting and producing single cells which divide to produce a mixture of small clumps and single cells (Figure 2C).10.7554/eLife.04875.005Figure 2.24-hour autonomous oscillations in dynamic aggregate assembly.


Evolving a 24-hr oscillator in budding yeast.

Wildenberg GA, Murray AW - Elife (2014)

Dynamics of oscillator aggregate assembly.(A) Low magnification images of synchronized E6-1 cultures that contained two subclones, one labeled with CFP (CFP+) and one without CFP (CFP−). Single cells that were selected as dim events by FACS and whose cell walls were labeled with Oregon green 488-X assemble a lineage-based clump and do not adhere to other cell lineages (i.e., no CFP (+/−) clumps) (left panels). At the peak multicellular phase, clumps were labeled with Oregon green 488-X and followed. These cells give rise to the single cell stage by fragmenting and producing non-adherent daughters so that the majority of the population is single celled by the end of the complete, 24-hr cycle (right panels). (B) Schematic of the mixing experiment performed by mixing, sonicating, and incubating asynchronous CFP+ and CFP− populations: cell separation defects will result in single colored clumps but adhesive cells will stick non-specifically to each other and produce clumps containing cells of both colors. Representative image of a single clump (white outline) showing E6-1 cells form clumps by adhering to each other.DOI:http://dx.doi.org/10.7554/eLife.04875.006
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4270073&req=5

fig2s1: Dynamics of oscillator aggregate assembly.(A) Low magnification images of synchronized E6-1 cultures that contained two subclones, one labeled with CFP (CFP+) and one without CFP (CFP−). Single cells that were selected as dim events by FACS and whose cell walls were labeled with Oregon green 488-X assemble a lineage-based clump and do not adhere to other cell lineages (i.e., no CFP (+/−) clumps) (left panels). At the peak multicellular phase, clumps were labeled with Oregon green 488-X and followed. These cells give rise to the single cell stage by fragmenting and producing non-adherent daughters so that the majority of the population is single celled by the end of the complete, 24-hr cycle (right panels). (B) Schematic of the mixing experiment performed by mixing, sonicating, and incubating asynchronous CFP+ and CFP− populations: cell separation defects will result in single colored clumps but adhesive cells will stick non-specifically to each other and produce clumps containing cells of both colors. Representative image of a single clump (white outline) showing E6-1 cells form clumps by adhering to each other.DOI:http://dx.doi.org/10.7554/eLife.04875.006
Mentions: To analyze the oscillations in cell association, we followed the behavior of E6-1 cells grown at low population density using two different labels. The first, cerulean fluorescent protein (CFP) was expressed from a constitutive promoter (PACT1) to distinguish two genetically identical subclones: one expressed CFP (CFP+) and the other did not (CFP−). The second was introduced by labeling cells, just after FACS selection, by covalently linking Oregon Green 488-X, a yellow fluorescent molecule, to their cell walls (Hoch et al., 2005). Because a daughter’s cell wall is entirely new, the original cells retain the yellow label and their daughters are unlabeled (Barral et al., 2000). We followed these cultures as they proliferated and eventually switched to the other phase of the morphological oscillation (Figure 2A,B and Figure 2—figure supplement 1A). Single cells that were selected for by sorting for low YFP fluorescence and covalently labeled (T = 0 hr), proliferated to become clumps composed of a single yellow cell surrounded with non-yellow cells that had the same CFP expression state as the original single cell. The absence of clumps containing more than one yellow cell or both CFP+ and CFP− cells in the same clump shows that cells emerging from the single-cell phase build lineage-based clumps. Consistent with this interpretation, we saw synchronous oscillations in cell association even at very high dilution (∼103 cells/ml). Multicellular clumps, which were covalently labeled at the time of peak aggregation gave rise to single cells by a combination of two mechanisms: fragmenting to produce smaller clumps and proliferating to produce single-celled offspring. Smaller clumps consisting of yellow cells and newly born non-yellow cells were observed 3 hr after (T = 13 hr) the peak aggregation time (10 hr). By 18 hr, the population was a mixture of medium-sized and smaller clumps and single cells, and by 24 hr non-yellow, single cells, and small clumps were the bulk of the population, representing the completion of one cycle. When unsynchronized CFP+ and CFP− subclones were sonicated to break up all the clumps and cultured together for 90 min, clumps that re-associated were composed of a mixture of CFP+ and CFP− cells showing that clumps form by cells sticking together rather than failing to separate after division (Figure 2—figure supplement 1B). These results show that cells oscillate between two states over a 24-hr period: single cells produce offspring that immediately stick to their mothers to assemble a lineage-based clump, which later produces a population of single cells and small clumps as a result of clumps fragmenting and producing single cells which divide to produce a mixture of small clumps and single cells (Figure 2C).10.7554/eLife.04875.005Figure 2.24-hour autonomous oscillations in dynamic aggregate assembly.

Bottom Line: We identified eight putative causative mutations in one clone and recreated the evolved phenotype in the ancestral strain.The mutated genes lack obvious relationships to each other, but multiple lineages change from the haploid to the diploid pattern of gene expression.We show that a novel, complex phenotype can evolve by small sets of mutations in genes whose molecular functions appear to be unrelated to each other.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, United States.

ABSTRACT
We asked how a new, complex trait evolves by selecting for diurnal oscillations in the budding yeast, Saccharomyces cerevisiae. We expressed yellow fluorescent protein (YFP) from a yeast promoter and selected for a regular alternation between low and high fluorescence over a 24-hr period. This selection produced changes in cell adhesion rather than YFP expression: clonal populations oscillated between single cells and multicellular clumps. The oscillations are not a response to environmental cues and continue for at least three cycles in a constant environment. We identified eight putative causative mutations in one clone and recreated the evolved phenotype in the ancestral strain. The mutated genes lack obvious relationships to each other, but multiple lineages change from the haploid to the diploid pattern of gene expression. We show that a novel, complex phenotype can evolve by small sets of mutations in genes whose molecular functions appear to be unrelated to each other.

No MeSH data available.


Related in: MedlinePlus